Inhibitory Effect of Paeonol on Vascular Smooth Muscle Cell Senescence by Reducing SIRT6/PARP1-mediated DNA Damage

JIANG Tingting; LIU Yarong; SHI Xiaoyan; YANG Yulong; DAI Min

doi:10.13422/j.cnki.syfjx.20230707

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Inhibitory Effect of Paeonol on Vascular Smooth Muscle Cell Senescence by Reducing SIRT6/PARP1-mediated DNA Damage

更新时间：2023-04-13

- Inhibitory Effect of Paeonol on Vascular Smooth Muscle Cell Senescence by Reducing SIRT6/PARP1-mediated DNA Damage
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 29, Issue 10, Pages: 83-92(2023)
- 作者机构：
  
  安徽中医药大学药学院，安徽省中药研究与开发重点实验室，合肥 230012
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20230707
  CLC： R2-0;R22;R285.5;R289;R33
- Published：20 May 2023，
  
  Published Online：19 March 2023，
  
  Received：29 November 2022，
- 稿件说明：
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蒋婷婷,刘雅蓉,施晓艳等.丹皮酚减轻SIRT6/PARP1介导的DNA损伤抑制血管平滑肌细胞衰老的作用[J].中国实验方剂学杂志,2023,29(10):83-92.

JIANG Tingting,LIU Yarong,SHI Xiaoyan,et al.Inhibitory Effect of Paeonol on Vascular Smooth Muscle Cell Senescence by Reducing SIRT6/PARP1-mediated DNA Damage[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(10):83-92.
蒋婷婷,刘雅蓉,施晓艳等.丹皮酚减轻SIRT6/PARP1介导的DNA损伤抑制血管平滑肌细胞衰老的作用[J].中国实验方剂学杂志,2023,29(10):83-92. DOI： 10.13422/j.cnki.syfjx.20230707.

JIANG Tingting,LIU Yarong,SHI Xiaoyan,et al.Inhibitory Effect of Paeonol on Vascular Smooth Muscle Cell Senescence by Reducing SIRT6/PARP1-mediated DNA Damage[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(10):83-92. DOI： 10.13422/j.cnki.syfjx.20230707.

摘要

目的

探讨丹皮酚（Pae）对血管紧张素Ⅱ（AngⅡ）诱导的血管平滑肌细胞（VSMCs）衰老的作用及对沉默调节信息因子6（SIRT6）/腺苷二磷酸核糖聚合酶1（PARP1）信号通路的影响。

方法

建立AngⅡ（100 nmol·L

-1

）诱导的VSMCs应激性衰老模型，实验分为正常组、模型组、Pae低、中、高（30、60、120 μmol·L

-1

）浓度组。采用

-半乳糖苷酶（SA-

-Gal）染色法检测细胞衰老阳性率；细胞增殖与活性检测（CCK-8）法检测细胞增殖能力；蛋白免疫印迹法（Western blot）检测SIRT6、PARP1、衰老相关基因p16、p21、p53、增殖细胞核抗原（PCNA）、脱氧核糖核酸（DNA）损伤蛋白磷酸化的组蛋白H2AX（即p-H2AX或

-H2AX）的表达并采用EdU染色法检测VSMCs增殖变化；siRNA-SIRT6转染制备沉默的VSMCs，观察SIRT6沉默的VSMCs中Pae对SIRT6、PARP1及p16、

-H2AX蛋白的表达。

结果

SA-

-Gal染色结果显示，与正常组比较，模型组SA-

-Gal染色衰老阳性率增加（

0.01）；与模型组比较，Pae给药组均有效降低SA-

-Gal染色阳性率（

0.05，

0.01）。Western blot结果显示，与正常组比较，模型组PCNA、SIRT6、PARP1表达下调、衰老相关蛋白p16、p21、p53、

-H2AX的表达上调（

0.05，

0.01）；与模型组比较，Pae给药干预后，各浓度组促进了PCNA、SIRT6、PARP1的蛋白表达，抑制了p16、p21、p53、

-H2AX的蛋白表达，且呈剂量依赖性（

0.05，

0.01）。EdU染色结果显示，与正常组比较，模型组EdU阳性细胞数减少（

0.01）；与模型组比较，Pae给药组EdU阳性细胞数明显增加（

0.05，

0.01）。SIRT6沉默后，Pae促进SIRT6、PARP1及抑制p16的作用被逆转（

0.05，

0.01）。此外加入SIRT6抑制剂IN-1可促进AngⅡ诱导细胞衰老的发生（

0.05，

0.01）。

结论

Pae能够有效抑制VSMCs的衰老，其机制作用可能与调控SIRT6/PARP1信号通路相关。

Abstract

Objective

To investigate whether the effects of paeonol （Pae） on angiotensin Ⅱ （AngⅡ）-induced senescence in vascular smooth muscle cells （VSMCs） were related to angiotensinogen of silencing regulatory information factor 6 （SIRT6）/adenosine diphosphate ribose polymerase 1 （PARP1） signaling pathway in VSMCs.

Method

The model of VSMC-stress aging induced by AngⅡ （100 nmol·L

-1

） was established. The rats were divided into normal group， model group， low， medium， and high-concentration Pae groups （30， 60， 120 μmol·L

-1

）. The positive rate of cell senescence was detected by SA-

-Gal staining， the ability of cell proliferation was detected by the cell counting kit-8 （CCK-8） method， the expression of SIRT6， PARP1， p16， p21， p53， proliferating cell nuclear antigen （PCNA）， deoxyribonucleic acid （DNA）-damaged protein

-H2AX was detected by Western blot， and VSMC proliferation was detected by EdU staining. The silenced VSMCs were prepared by siRNA-SIRT6 transfection， and the protein expressions of SIRT6， PARP1， p16， and

-H2AX in VSMCs silenced by SIRT6 were observed.

Result

The results of SA-

-Gal staining showed that the senescence positive rate of SA-

-Gal staining in the model group was higher than that in the normal group （

0.01）， and the positive rate of SA-

-Gal staining in the Pae group was significantly lower than that in the model group （

0.05，

0.01）. The results of Western blot showed that as compared with the normal group， the expression of PCNA， SIRT6， and PARP1 in the model group was down-regulated， and the expression of aging-related proteins p16， p21， p53， and

-H2AX was up-regulated in the model group （

0.05，

0.01）. Compared with the model group， Pae promoted the protein expression of PCNA， SIRT6， and PARP1 and inhibited the protein expression of p16， p21， p53， and

-H2AX in a dose-dependent manner （

0.05，

0.01）. The results of EdU staining showed that the number of EdU positive cells in the model group was lower than that in the normal group （

0.01）， and the number of EdU positive cells in Pae groups was significantly higher than that in the model group （

0.05，

0.01）. After SIRT6 silencing， the effects of Pae on promoting SIRT6 and PARP1 and inhibiting P16 were reversed （

0.05，

0.01）. In addition， the addition of SIRT6 inhibitor （IN-1） promoted the occurrence of cell senescence induced by AngⅡ （

0.05，

0.01）.

Conclusion

Pae can effectively inhibit the aging of VSMCs， and its mechanism may be related to the regulation of SIRT6/PARP1 signal pathway.

关键词

丹皮酚沉默调节信息因子6（SIRT6）腺苷二磷酸核糖聚合酶1（PARP1）血管平滑肌细胞（VSMCs）衰老

Keywords

paeonolsilencing regulatory information factor 6 （SIRT6）adenosine diphosphate ribose polymerase 1 （PARP1）vascular smooth muscle cells （VSMCs）senescence

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